We are in the midst of an epidemic of obesity and type 2 diabetes. The discovery of new pathways of energy metabolism is critically needed to address this pressing medical problem. Using untargeted metabolomics, we have identified a new pathway of energy expenditure mediated by family of bioactive lipids called N-acyl amino acids. Certain N-acyl amino acids and stimulate mitochondrial respiration by promoting proton leak. We have also de-orphanized a novel upstream enzyme, PM20D1 (peptidase M20 domain containing 1), that functions as an extracellular N-acyl amino acid synthase/hydrolase. Pharmacological or genetic elevation of circulating N-acyl amino acids increases energy expenditure, reduces adiposity, and improves glucose homeostasis in mouse models of diet-induced obesity. However, we are still early in our understanding of N-acyl amino acids. What remains unknown is how N-acyl amino acids promote proton conductance across the inner mitochondrial membrane, what other extracellular mechanisms regulate N- acyl amino acid levels, and whether this pathway could be useful for the treatment of obesity-associated disorders. Answers are critically needed to understand the biology and therapeutic potential of this energy expenditure pathway in metabolic disease. The long-term goal of this project is to harness energy expenditure pathways for the treatment of obesity and type 2 diabetes. The overall objective of this proposal is to mechanistically dissect the regulators of the N-acyl amino acid pathway and to assess the therapeutic potential of these bioactive lipids. Our central hypothesis is that N-acyl amino acid bioactivity is regulated by both intracellular and extracellular proteins, and that this pathway can be pharmacologically leveraged for the treatment of obesity and type 2 diabetes. We will test this hypothesis via three Specific Aims: 1) Determine how N-acyl amino acids stimulate uncoupled respiration; 2) Determine the mechanisms that control circulating N-acyl amino acid levels; and 3) Evaluate the bioactivity of synthetic N-acyl amino acid analogs in diet-induced obesity mouse models. Successful completion of this proposal will provide a detailed, mechanistic understanding of the regulation and function of N-acyl amino acids in energy metabolism, as well as a pharmacological evaluation of this pathway for the treatment of obesity-associated dis- eases such as type 2 diabetes.